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NIP1;1, an Aquaporin Homolog, Determines the Arsenite Sensitivity of Arabidopsis thaliana

2008; Elsevier BV; Volume: 284; Issue: 4 Linguagem: Inglês

10.1074/jbc.m806881200

1083-351X

Takehiro Kamiya, M. Tanaka, Namiki Mitani, Jian Feng, Masayoshi Maeshima, Toru Fujiwara,

Aluminum toxicity and tolerance in plants and animals

Arsenite [As(III)] is highly toxic to organisms, including plants. Very recently, transporters in rice responsible for As(III) transport have been described (Ma, J. F., Yamaji, N., Mitani, N., Xu, X. Y., Su, Y. H., McGrath, S. P., and Zhao, F. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 9931–9935), but little is known about As(III) tolerance. In this study, three independent As(III)-tolerant mutants were isolated from ethyl methanesulfonate-mutagenized M2 seeds of Arabidopsis thaliana. All three mutants carried independent mutations in Nodulin 26-like intrinsic protein 1;1 (NIP1;1), a homolog of an aquaporin. Two independent transgenic lines carrying T-DNA in NIP1;1 were highly tolerant to As(III), establishing that NIP1;1 is the causal gene of As(III) tolerance. Because an aquaglyceroporin is able to transport As(III), we measured As(III) transport activity. When expressed in Xenopus oocytes, NIP1;1 was capable of transporting As(III). As content in the mutant plants was 30% lower than in wild-type plants. Promoter β-glucuronidase and real-time PCR analysis showed that NIP1;1 is highly expressed in roots, and GFP-NIP1;1 is localized to the plasma membrane. These data show that NIP1;1 is involved in As(III) uptake into roots and that disruption of NIP1;1 function confers As(III) tolerance to plants. NIP1;2 and NIP5;1, closely related homologs of NIP1;1, were also permeable to As(III). Although the disruption of these genes reduced the As content in plants, As(III) tolerance was not observed in nip1;2 and nip5;1 mutants. This indicates that As(III) tolerance cannot be simply explained by decreased As contents in plants. Arsenite [As(III)] is highly toxic to organisms, including plants. Very recently, transporters in rice responsible for As(III) transport have been described (Ma, J. F., Yamaji, N., Mitani, N., Xu, X. Y., Su, Y. H., McGrath, S. P., and Zhao, F. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 9931–9935), but little is known about As(III) tolerance. In this study, three independent As(III)-tolerant mutants were isolated from ethyl methanesulfonate-mutagenized M2 seeds of Arabidopsis thaliana. All three mutants carried independent mutations in Nodulin 26-like intrinsic protein 1;1 (NIP1;1), a homolog of an aquaporin. Two independent transgenic lines carrying T-DNA in NIP1;1 were highly tolerant to As(III), establishing that NIP1;1 is the causal gene of As(III) tolerance. Because an aquaglyceroporin is able to transport As(III), we measured As(III) transport activity. When expressed in Xenopus oocytes, NIP1;1 was capable of transporting As(III). As content in the mutant plants was 30% lower than in wild-type plants. Promoter β-glucuronidase and real-time PCR analysis showed that NIP1;1 is highly expressed in roots, and GFP-NIP1;1 is localized to the plasma membrane. These data show that NIP1;1 is involved in As(III) uptake into roots and that disruption of NIP1;1 function confers As(III) tolerance to plants. NIP1;2 and NIP5;1, closely related homologs of NIP1;1, were also permeable to As(III). Although the disruption of these genes reduced the As content in plants, As(III) tolerance was not observed in nip1;2 and nip5;1 mutants. This indicates that As(III) tolerance cannot be simply explained by decreased As contents in plants. Arsenic (As) 2The abbreviations used are: As, arsenic; GFP, green fluorescent protein; NIP1;1, Nodulin 26-like intrinsic protein 1;1; MES, 4-morpholineethanesulfonic acid; EMS, ethyl methanesulfonate; ICP-MS, inductively coupled plasma mass spectrometry. is a very toxic but not essential element for most organisms except for arsenate-reducing bacteria (1Oremland R.S. Stolz J.F. Science.. 2003; 300: 939-944Google Scholar). Arsenic represents a major environmental contaminant in several regions of the world. Long-term exposure to As causes skin diseases and cancers in humans. In most As-contaminated areas, As ingested by humans is derived from groundwater that is naturally contaminated with As. Both drinking water and irrigation with As-contaminated groundwater contribute to human ingestion (2British Geological Survey Kinniburgh D.G. Smedley P.L. Arsenic Contamination of Groundwater in Bangladesh. 1-4. British Geological Survey, Keyworth, UK2001Google Scholar, 3Chowdhury U.K. Biswas B.K. Chowdhury T.R. Samanta G. Mandal B.K. Basu G.C. Chanda C.R. Lodth D. Saha K.C. Mukherjee S.K. Roy S. Kabir S. Quamruzzaman Q. Chakraborti D. Environ. Health Perspect.. 2000; 108: 393-397Google Scholar). This is especially serious in West Bengal, India, and Bangladesh. Bangladesh has the most serious problem in terms of both the number of people affected and the severity of health problems (2British Geological Survey Kinniburgh D.G. Smedley P.L. Arsenic Contamination of Groundwater in Bangladesh. 1-4. British Geological Survey, Keyworth, UK2001Google Scholar, 3Chowdhury U.K. Biswas B.K. Chowdhury T.R. Samanta G. Mandal B.K. Basu G.C. Chanda C.R. Lodth D. Saha K.C. Mukherjee S.K. Roy S. Kabir S. Quamruzzaman Q. Chakraborti D. Environ. Health Perspect.. 2000; 108: 393-397Google Scholar). The major ingestion pathway of As is drinking As-contaminated water, followed by eating foods grown with contaminated water. Crops cultivated with As-containing groundwater accumulate As in their edible parts (4Meharg A.A. Trends Plant Sci.. 2004; 9: 415-417Google Scholar, 5Tripathi R.D. Srivastava S. Mishra S. Singh N. Tuli R. Gupta D.K. Maathuis F.J. Trends Biotechnol.. 2007; 25: 158-165Google Scholar). Rice is the major crop in these areas and is known to accumulate high levels of As in its grains (4Meharg A.A. Trends Plant Sci.. 2004; 9: 415-417Google Scholar). In addition to health effects, As inhibits the growth of rice, which leads to a reduction in yield (6Marin A.R. Masscheleyn P.H. Patrick Jr., W.H. Plant Soil.. 1992; 139: 175-183Google Scholar, 7Rahman M.A. Hasegawa H. Rahman M.M. Islam M.N. Miah M.A.M. Tasmin A. Chemosphere.. 2007; 67: 1072-1079Google Scholar). Both reducing As uptake and generation of As-tolerant plants, especially rice, are useful methods to circumvent problems associated with As contamination. In the environment, both inorganic and organic As are present, and the inorganic form is more toxic. Two major inorganic As species in the environment are known: arsenate [As(V)] and arsenite [As(III)]. Transport processes of inorganic As have been studied in a number of organisms, and those involved in As uptake have been identified in bacteria, yeast, and animals (8Rosen B.P. FEBS Lett.. 2002; 529: 86-92Google Scholar). As(V) is taken up via phosphate transport systems, because As(V) (AsO3–4) is an analog of phosphate. The As(III) molecule is uncharged at neutral pH, and it enters the cell via aquaglyceroporins, which belong to the major intrinsic protein (MIP) family. Sanders et al. (9Sanders O.I. Rensing C. Kuroda M. Mitra B. Rosen B.P. J Bacteriol.. 1997; 179: 3365-3367Google Scholar) first identified GlpF as an As(III) transporter by screening random-mutagenized Escherichia coli for antimonite [Sb(III)]-tolerant mutants. Sb(III), which is a congener of As(III), is also transported by aquaglyceroporins. Subsequently, Fps1p in yeast, AQP7 and AQP9 in humans, and LmAQP1 in Leishmania major have been identified as As(III) transporters (10Wysocki R. Chéry C.C. Wawrzycka D. Van Hulle M. Cornelis R. Thevelein J.M. Tamás M.J. Mol Microbiol.. 2001; 40: 1391-1401Google Scholar, 11Liu Z. Shen J. Carbrey J.M. Mukhopadhyay R. Agre P. Rosen B.P. Proc. Natl. Acad. Sci. U. S. A.. 2002; 99: 6053-6058Google Scholar, 12Figarella K. Uzcategui N.L. Zhou Y. LeFurgey A. Ouellette M. Bhattacharjee H. Mukhopadhyay R. Mol. Microbiol.. 2007; 65: 1006-1017Google Scholar). In addition to the MIP family, hexose permeases are also involved in As(III) uptake in yeast (13Liu Z. Boles E. Rosen B.P. J. Biol. Chem.. 2004; 279: 17312-17318Google Scholar). In plants, a number of studies have demonstrated the physiological properties and molecular mechanisms of inorganic As uptake. In rice, uptake kinetics of As(V) and As(III) follow the Michaelis-Menten equation (14Abedin M.J. Feldmann J. Meharg A.A. Plant Physiol.. 2002; 128: 1120-1128Google Scholar), suggesting the presence of a transporter. In Arabidopsis thaliana, Pho1;1 and Pho1;4 are responsible for As(V) uptake (15Shin H. Shin H.S. Dewbre G.R. Harrison M.J. Plant J.. 2004; 39: 629-642Google Scholar). In the case of As(III), glycerol and Sb(III) inhibit As(III) uptake into rice roots (16Meharg A.A. Jardine L. New Phytol.. 2003; 157: 39-44Google Scholar). Because glycerol and Sb(III) are substrates for aquaglyceroporin, the finding suggests the involvement of aquaglyceroporins in As(III) uptake in plants. Very recently, involvement of aquaglyceroporins, including OsNIP2;1, have been reported in rice (17Ma J.F. Yamaji N. Mitani N. Xu X.Y. Su Y.H. McGrath S.P. Zhao F.J. Proc. Natl. Acad. Sci. U. S. A.. 2008; 105: 9931-9935Google Scholar). In addition, in A. thaliana, NIP5;1, NIP6;1, and NIP7;1 are also permeable to As(III) in the yeast expression system (18Bienert G.P. Thorsen M. Schüssler M.D. Nilsson H.R. Wagner A. Tamás M.J. Jahn T.P. BMC Biol.. 2008; 6: 26Google Scholar, 19Isayenkov S.V. Maathuis F.J. FEBS Lett.. 2008; 14: 1625-1628Google Scholar). Considering that these transporters are involved in toxic As uptake into plants, disruption of these transporters is expected to confer As tolerance, as is the case in Escherichia coli and yeast. Disruption of phosphate transporters and NIP7;1 confer the As(V) tolerance and moderate As(III) tolerance to A. thaliana, respectively (15Shin H. Shin H.S. Dewbre G.R. Harrison M.J. Plant J.. 2004; 39: 629-642Google Scholar, 19Isayenkov S.V. Maathuis F.J. FEBS Lett.. 2008; 14: 1625-1628Google Scholar). After As enters the cell, As(V) is reduced to As(III) by a reductase. As(III) is excluded from the cell by ArsAB and Acr3p transporters in E. coli and yeast, respectively (8Rosen B.P. FEBS Lett.. 2002; 529: 86-92Google Scholar). In addition to exclusion, yeast has Ycf1p, an ABC transporter, which sequesters the As-glutathione complex into the vacuole (8Rosen B.P. FEBS Lett.. 2002; 529: 86-92Google Scholar). In plants, As-phytochelatin and As-glutathione complexes are found in As-treated plants (20Raab A. Feldmann J. Meharg A.A. Plant Physiol.. 2004; 134: 1113-1122Google Scholar), and the involvement of these complexes in As tolerance is being investigated. Because the gene conferring moderate levels of As(III) tolerance to plants has been reported, we used a forward genetic approach to identify the gene(s) that confer strong tolerance to As(III) in A. thaliana. We isolated three As(III)-tolerant mutants by screening ethyl methanesulfonate (EMS)-mutagenized A. thaliana plants and identified a gene responsible for tolerance to As(III). Plant Growth Conditions and Media—A. thaliana ecotype Col-0 was obtained from laboratory stock and was used for the physiological experiments. The seeds were surface-sterilized with bleach and sown onto half-strength Murashige-Skoog (MS) medium solidified with 1.2% Gellan Gum (Wako, Osaka, Japan) supplemented with 1% sucrose. The pH of the medium was adjusted with 0.05% MES and KOH to 5.7. For the arsenite [As(III)] treatment, arsenite trioxide (Wako) was added to the medium at the concentrations shown in individual experiments. The screening for As(III)-tolerant mutants was performed as described below. The EMS-mutagenized seeds (Col-0 gl1-1) were purchased from Lehle Seeds (Round Rock, TX). The seeds were surface-sterilized and sown on half-strength MS medium containing 15 μm As(III). After incubation for 2 days at 4 °C, the plates were placed vertically, and the plants were grown at 22 °C for 10 days under a 16-h light/8-h dark photoperiod. Plants with longer roots compared with the wild type were selected, and the mutant phenotype was confirmed in the M3 generation. Transgenic Plants Carrying T-DNA in NIPs—Transgenic plants carrying T-DNA were obtained from the Arabidopsis Biological Resource Center (ABRC) at Ohio State University. T-DNA homozygous plants were selected using primers specific to NIP1;1, NIP1;2. For NIP1;1 mutants, SALK_016617 and SALK_017916 were named nip1;1-1 and nip1;1-2, respectively. For NIP1;2, SALK_046418 and SALK_126593 were named nip1;2-1 and nip1;2-2, respectively. nip5;1-1 was reported previously (21Takano J. Wada M. Ludewig U. Schaaf G. von Wirén N. Fujiwara T. Plant Cell.. 2006; 18: 1498-1509Google Scholar). Preparation of Total RNA and Real-time PCR Analysis—Total RNA was prepared from plants with an RNeasy Plant Mini kit (Qiagen, Valencia, CA), and DNase treatment was performed with an RNase-free DNase set (Qiagen). RNA was converted into cDNA using the PrimeScript RT reagent kit (Takara, Ohtsu, Japan). The cDNA was diluted 10-fold and used for real-time PCR analysis with Dice (Takara) using SYBR Premix Ex TaqII (Takara). The primer sequences used for NIP1;1 were as follows; 5′-GCCAACTCTTGGTGCGATTG-3′ and 5′-GTGCTACCGATTCTCACGGTC-3′. The primer sequences used for Elongation factor 1α (internal standard) were described in a previous report (21Takano J. Wada M. Ludewig U. Schaaf G. von Wirén N. Fujiwara T. Plant Cell.. 2006; 18: 1498-1509Google Scholar). As(III) Transport Assay in Xenopus Oocytes—To synthesize the cRNA, NIP1;1 cDNA was amplified by PCR using 5′-GACTAGTAGATGGCGGATATCTCGGGAAACG-3′ and 5′-GGAATTCTCAAGTGCTACCGATTCTCACGGTC-3′ (SpeI and EcoRI recognition sites are underlined). The resulting DNA fragment was digested with SpeI and EcoRI, and inserted into pOO2 (22Ludewig U. von Wirén N. Frommer W.B. J. Biol. Chem.. 2002; 277: 13548-13555Google Scholar) at the SpeI-EcoRI site. The cRNA was synthesized with the mMESSAGE mMACHINE SP6 kit (Ambion, Austin, TX) and purified with a MEGA clear column (Ambion). The influx transport was measured as described previously (17Ma J.F. Yamaji N. Mitani N. Xu X.Y. Su Y.H. McGrath S.P. Zhao F.J. Proc. Natl. Acad. Sci. U. S. A.. 2008; 105: 9931-9935Google Scholar). Briefly, 50 nl of cRNA solution (containing 25 ng of cRNA) or water were injected into Xenopus laevis oocytes. After incubation with modified Barth's saline (MBS) for 24 h, six oocytes were incubated with MBS containing 100 μm As(III) for the indicated periods. At the end of exposure, the oocytes were washed with ice-cold MBS four times and subjected to As determination with ICP-MS as described below. As Accumulation in Plants—Seeds were sown on the half-strength MS medium containing 5 μm As(III). After incubation for 2 days at 4 °C, the plates were placed vertically, and the plants were grown at 22 °C for 7 days under a 16-h light/8-h dark photoperiod. The plants were washed with distilled water three times, dried at 65 °C for more than 2 days, and subjected to As determination with ICP-MS as described below. Determination of As Concentration using ICP-MS—The samples, including oocytes and dried plants, were digested with concentrated HNO3 at 110 °C. After complete digestion, the sample was dissolved in 0.08 n HNO3 containing 10 ppb Ge. 72Ge was used as an internal standard. The mass 75 was monitored as the As signal. To remove the contribution from 40Ar35Cl, 77Se and 82Se were simultaneously monitored and the actual As signal was calculated with the equation given in the EPA 200.8 method (23US EPA Methods for the Determination of Metals in Environmental Samples. US Environmental Protection Agency, Cincinnati, Ohio1992Google Scholar). Promoter-GUS Analysis—The DNA fragment corresponding to the promoter region of NIP1;1 was amplified with PCR using genomic DNA as template and the primers 5′-CGATTTCCCGTGATCCGTGGCACC-3′ and 5′-GCGTCGACTCCCGAGATATCCGCCATAAGTGAC-3′ (SalI recognition site underlined). The amplified DNA fragment was digested with SalI, inserted into pENTR2B at the XmnI-SalI sites, and then transferred to the destination vector pMDC139 (24Curtis M.D. Grossniklaus U. Plant Physiol.. 2003; 133: 462-469Google Scholar) using LR clonase (Invitrogen). Col-0 plants were transformed with the Agrobacterium-mediated floral dip method. GUS staining was performed as described previously (25Kamiya T. Akahori T. Ashikari M. Maeshima M. Plant Cell Physiol.. 2006; 47: 96-106Google Scholar). Determination of Subcellular Localization using GFP—A GFP-NIP1;1 fusion gene was constructed as described below. NIP1;1 was amplified by PCR using the primers 5′-GGAATTCATGGCGGATATCTCGGG-3′ and 5′-GAGAGTCGACTCAAGTGCTACCGATTCTCA-3′ (underlines indicate EcoRI and SalI recognition sequences, respectively). The resulting DNA fragment was digested with EcoRI and SalI and inserted into pENTR2B (Invitrogen, Tokyo, Japan) at the EcoRI-XhoI sites and then transferred to the binary vector pMDC45 (24Curtis M.D. Grossniklaus U. Plant Physiol.. 2003; 133: 462-469Google Scholar) using LR clonase (Invitrogen). nip1;1-1 plants were transformed with the Agrobacterium tumefaciens-mediated floral dip method. After selection with hygromycin, GFP fluorescence of resistant plants was observed with fluorescence microscopy in the T1 generation to select the fluorescence-emitting transgenic plants. The T2 generation was used for fluorescence observations using confocal laser-scanning microscopy. Screening for As(III)-tolerant Mutants—To obtain insights into As(III) tolerance mechanisms, the EMS-mutagenized M2 population of A. thaliana was screened for As(III)-tolerant mutants. We chose 15 μm As(III) as the selection condition. At this concentration, the root length of the wild type was 20% of that of plants growing on the As(III) minus medium after a 10-day incubation. Approximately 30,000 M2 seeds were sown onto half-strength MS medium containing 15 μm As(III) and grown vertically for 10 days. Plants with roots more than two times longer than the wild type were selected. After confirmation of the phenotype in the M3 generation, three independent mutants, 7-1, 9-1, and 10-1, were obtained (Fig. 1A). These were derived from independent batches of EMS-mutagenized M2 populations. These mutant roots were more than three times longer than those of the wild type in the presence of 10, 15, and 30 μm As(III), at which concentration the growth of wild-type roots was strongly inhibited (Fig. 1B). The F2 population from crosses between Ler and 7-1 mutants was sown onto medium containing 15 μm As(III) and segregated into tolerant, weakly tolerant, and sensitive phenotypes at a ratio of 1:2:1 (Fig. 1C). The same phenotype was observed in the populations of 9-1 and 10-1 (data not shown). These results indicate that the As(III)-tolerant phenotype is caused by a single mutation, and the mutated alleles are all semidominant. Identification of NIP1;1 as a Causal Gene for As(III) Tolerance—We speculated that aquaglyceroporins might be involved in As(III) tolerance based on previous studies (9Sanders O.I. Rensing C. Kuroda M. Mitra B. Rosen B.P. J Bacteriol.. 1997; 179: 3365-3367Google Scholar, 10Wysocki R. Chéry C.C. Wawrzycka D. Van Hulle M. Cornelis R. Thevelein J.M. Tamás M.J. Mol Microbiol.. 2001; 40: 1391-1401Google Scholar, 11Liu Z. Shen J. Carbrey J.M. Mukhopadhyay R. Agre P. Rosen B.P. Proc. Natl. Acad. Sci. U. S. A.. 2002; 99: 6053-6058Google Scholar, 12Figarella K. Uzcategui N.L. Zhou Y. LeFurgey A. Ouellette M. Bhattacharjee H. Mukhopadhyay R. Mol. Microbiol.. 2007; 65: 1006-1017Google Scholar, 17Ma J.F. Yamaji N. Mitani N. Xu X.Y. Su Y.H. McGrath S.P. Zhao F.J. Proc. Natl. Acad. Sci. U. S. A.. 2008; 105: 9931-9935Google Scholar, 19Isayenkov S.V. Maathuis F.J. FEBS Lett.. 2008; 14: 1625-1628Google Scholar). Among the MIP family in plants, some members of nodulin 26-like intrinsic proteins (NIPs), which are classified as aquaglyceroporins in plants (26Zardoya R. Ding X. Kitagawa Y. Chrispeels M.J. Proc. Natl. Acad. Sci. U. S. A.. 2002; 99: 14893-14896Google Scholar), were shown to transport small neutral molecules including boric acid and silicic acid (21Takano J. Wada M. Ludewig U. Schaaf G. von Wirén N. Fujiwara T. Plant Cell.. 2006; 18: 1498-1509Google Scholar, 27Ma J.F. Tamai K. Yamaji N. Mitani N. Konishi S. Katsuhara M. Ishiguro M. Murata Y. Yano M. Nature.. 2006; 440: 688-691Google Scholar). As(III) is also a small neutral molecule, and we speculated that As(III) tolerance may be due to mutation(s) in a NIP gene. In the A. thaliana genome, there are nine NIP genes (28Johanson U. Karlsson M. Johansson I. Gustavsson S. Sjövall S. Fraysse L. Weig A.R. Kjellbom P. Plant Physiol.. 2001; 126: 1358-1369Google Scholar), and we obtained seven T-DNA insertion mutants of NIPs(NIP1;1, NIP1;2, NIP3;1, NIP4;1, NIP5;1, NIP6;1, and NIP7;1) and examined their As(III) tolerance. Among NIP mutants examined, only NIP1;1 T-DNA insertion mutants (nip1;1-1, nip1;1-2) exhibited high tolerance to 15 μm As(III) (Fig. 2A, supplemental Fig. S1). In contrast, transgenic plants carrying T-DNA in NIP1;2, which is the most similar to NIP1;1 in terms of amino acid sequences among the NIP family members, did not show As(III) tolerance. Similarly, transgenic plants carrying T-DNA in NIP5;1, which mediates boric acid uptake from soil (21Takano J. Wada M. Ludewig U. Schaaf G. von Wirén N. Fujiwara T. Plant Cell.. 2006; 18: 1498-1509Google Scholar), also did not show As(III) tolerance (Fig. 2A). Furthermore, NIP7;1, which confers As(III) tolerance (19Isayenkov S.V. Maathuis F.J. FEBS Lett.. 2008; 14: 1625-1628Google Scholar) did not confer strong tolerance in our experimental conditions (supplemental Fig. S1). As shown in Fig. 2B, nip1;1-1 and nip1;1-2 have T-DNA insertions in the third intron and the fifth exon, respectively. The mRNA accumulation level was very low or not detected in nip1; 1-1 and nip1;1-2, respectively, indicating that nip1;1-1 and nip1;1-2 are very strong and null alleles, respectively (Fig. 2C). Because nip1;1 showed high tolerance to As(III), NIP1;1 genomic sequences of the As(III)-tolerant mutants 7-1, 9-1, and 10-1 were determined. All of the mutants had a single nucleotide substitution in NIP1;1 (Fig. 2B). Lines 7-1 and 9-1 mutants had mutations in the third and second exons, respectively. These mutations caused amino acid substitutions in the proteins Gly63 to Glu63 and Thr188 to Ile188, respectively. The line 10-1 mutant had a mutation in the fourth exon/intron junction, which resulted in a larger transcript than the wild type (Fig. 2D). The sequence analysis of the RT-PCR product of 10-1 indicated that the third intron was not spliced out (data not shown). The third intron contains an in-frame stop codon, and the NIP1;1 transcript of the 10-1 line is likely to produce a short and abnormal protein. To further confirm that NIP1;1 is the causal gene of As(III) tolerance, mapping analysis was carried out using F2 population from crosses between Ler and As(III) tolerant mutants. Markers on chromosome 4 showed the strong linkage to As(III) tolerance phenotype, and the mutation was located between markers F24G24 and F9D16 (supplemental Fig. S2). NIP1;1 is located in this interval, further supporting that NIP1;1 is the causal gene of the phenotype. NIP1;1 transcript accumulation was at a similar level among Col-0, 7-1, and 9-1 (Fig. 2E). In 10-1, NIP1;1 transcript accumulation was reduced to 25% of that of Col-0 (Fig. 2E). Taken together, these results establish that NIP1;1 is the causal gene for As(III)-tolerant mutants, and disruption of NIP1;1 function makes the plant tolerant to As(III). NIPs Transport As(III)—The fact that disruption of NIP1;1 confers strong As(III) tolerance suggests that NIP1;1 is an As(III) transporter that mediates toxic As(III) uptake into roots. To directly confirm the As(III) transport activity of the protein, we used a X. laevis oocyte expression system. A time course experiment showed that oocytes expressing NIP1;1 accumulated several times more As(III) than the control (Fig. 3A), demonstrating that NIP1;1 is capable of transporting As(III). We also measured the transport activity of the mutant proteins corresponding to 7-1, 9-1, and 10-1 lines (Fig. 3B). The content of As(III) in oocytes expressing mutant proteins was similar to that of the water-injected oocytes, suggesting that all mutant proteins [7-1 (G63E), 9-1 (T188I), and 10-1] lost transport activity (Fig. 3B), and that Gly63 and Thr188 are important amino acid residues for the protein to function as an As(III) transporter. The lack of transport activity of the mutant alleles of NIP1;1 corresponded with the As(III) tolerance phenotype of the mutant plants (Fig. 1). We also measured the transport activity of NIP1;2 and NIP5;1 in oocytes (Fig. 3B). As(III) transport activity was detected in both proteins. As Concentrations Were Reduced in nips Mutants—To examine the transport activity of NIPs in vivo, we measured As concentrations in the nip mutants. The mutant plants were grown vertically on plates containing 5 μm As(III) for 7 days, and As content in whole seedlings was determined. As shown in Fig. 3C, As concentrations in the nip1;1, nip1;2, and nip5;1 mutant plants were significantly lower than those in Col-0 plants. Taken together with the oocyte experiment, NIPs is active in As(III) transport in plants. Tissue Specificity of NIP1;1 Expression—To identify tissue specificity of expression in detail, a NIP1;1 promoter region (–2317 bp to +18 from the first ATG) was fused in-frame with the GUS (β-glucuronidase) gene. Four independent transgenic lines were generated, and GUS staining patterns were observed in the T2 generation. GUS staining was observed both in shoots and roots. The staining in leaves was observed mostly in stomata (Fig. 4, A and B). The root-hypocotyl junction, especially in the root region, showed strong GUS activity. In roots, the patterns differed between primary and lateral roots. In lateral roots, both root tips and steles were stained, whereas in primary roots, steles but not root tips were stained (Fig. 4, D and E). The pattern was consistent among the four lines. We also determined mRNA accumulation in shoots and roots using real-time PCR. The NIP1;1 mRNA in roots was 20 times higher than that in shoots (Fig. 4F). Subcellular Localization of NIP1;1—To identify the subcellular localization of NIP1;1, GFP-NIP1;1 fusion protein was expressed in the nip1;1-1 mutant under the cauliflower mosaic virus 35S-RNA promoter. About 50 independent lines were generated, and among these, GFP fluorescence was observed in 15 lines. These lines were subjected to the As(III) tolerance assay. Ten lines showed As(III)-sensitive phenotypes, and two of them (Nos. 7 and 8) were used (Fig. 5E). Sensitive phenotypes of the transgenic lines imply a complementation of the nip1;1-1 mutant, suggesting that GFP-NIP1;1 in these lines was functional (Fig. 5E). We also generated the transgenic plants carrying GFP-NIP1;1 construct as well as plants carrying NIP1;1-GFP construct under the control of the native NIP1;1 promoter. However, no fluorescence was observed after checking more than 20 lines for each constructs (data not shown). Subcellular localization of GFP fluorescence was observed with a confocal laser-scanning microscope, and two lines showed a similar fluorescence pattern (data not shown). In root tips, GFP fluorescence was observed in the cell periphery (Fig. 5, A and B). This fluorescence pattern is different from that of tonoplast-localized proteins (29Blaudez D. Kohler A. Martin F. Sanders D. Chalot M. Plant Cell.. 2003; 15: 2911-2928Google Scholar, 30Kataoka T. Watanabe-Takahashi A. Hayashi N. Ohnishi M. Mimura T. Buchner P. Hawkesford M.J. Yamaya T. Takahashi H. Plant Cell.. 2004; 16: 2693-2704Google Scholar). Vacuoles in cells near the root tips are observed as several vesicles. In the case of GFP-NIP1;1, we never observed such vesicle patterns, suggesting that GFP-NIP1;1 is localized to the plasma membrane. After plasmolysis treatment, the fluorescence mostly associated with plasma membrane, not with the cell walls (Fig. 5, C and D), further suggesting that GFP-NIP1;1 is localized to plasma membrane. Interestingly, the fluorescence was observed at the distal side of the epidermal cells (Fig. 5, A and B, inset). Furthermore, GFP fluorescence was observed only in outer cell layers, even though it is driven by the 35S promoter, which is active in all cell layers in roots. It is likely that NIP1;1 transcript accumulation is differentially and post-transcriptionally regulated in a cell type-dependent manner. In the present study, we demonstrated that NIP1;1 is the key determinant of As(III) tolerance in A. thaliana. We isolated three independent As(III) tolerant mutants in terms of root elongation by screening of 30,000 EMS-mutagenized M2 plants. All of them had mutations in NIP1;1 (Figs. 1 and 2). Moreover, nip1;1 mutant plants were highly tolerant to As(III), suggesting that NIP1;1 is the key determinant of of As(III) tolerance. Although it has been reported that nip7;1 mutants are moderately tolerant to As(III) (19Isayenkov S.V. Maathuis F.J. FEBS Lett.. 2008; 14: 1625-1628Google Scholar), the level of As(III) tolerance of nip1;1 mutant plants is much higher than that of nip7;1 under our experimental conditions (Fig. 2A and supplemental Fig. S1), suggesting that NIP1;1 is dominant over NIP7;1 in the determination of As(III) tolerance in A. thaliana. NIP1;1 is able to transport As(III) (Fig. 3, A and B) and is localized to the plasma membrane in roots (Fig. 5). These data strongly suggest that NIP1;1 is involved in As(III) uptake from the medium into the roots. However, As(III) content in nip1;1 mutant plants was reduced to 70% of that of wild-type plants (Fig. 3C). It is possible that As(III) transport systems other than NIP1;1 account for 70% of As(III) uptake into roots. NIP1;2 and NIP5;1 are among the candidate genes for As(III) uptake based on the result of oocyte experiment (Fig. 3B). In addition, two molecular biological analyses have recently demonstrated that A. thaliana NIPs are involved in As(III) transport. One is by Bienert et al. (18Bienert G.P. Thorsen M. Schüssler M.D. Nilsson H.R. Wagner A. Tamás M.J. Jahn T.P. BMC Biol.. 2008; 6: 26Google Scholar), who showed that NIP5;1 and NIP6;1 are able to transport As(III) and Sb(III) using a yeast expression system. The other is by Isayenkov and Maathuis (19Isayenkov S.V. Maathuis F.J. FEBS Lett.. 2008; 14: 1625-1628Google Scholar), who showed that T-DNA mutants of NIP7;1 are moderately tolerant to As(III) and have a reduced As(III) contents compared with wild type, and that NIP7;1 has As(III) transport activity in yeast. Taken together, NIP1;2, NIP5;1, NIP6;1, and NIP7;1, are major candidates for As(III) transport systems in nip1;1 mutant plants. In the present study, we determined the As(III) concentrations in nip1;2 and nip5;1 mutant plants. As shown in Fig. 3C, disruption of NIP1;2 and NIP5;1 resulted in 10–15% reduction of As in plants, suggesting that these two genes are likely candidates for As(III) transport systems in nip1;1 mutant plants. Alternatively, the modest reduction in As content in NIP1;1 mutant plants can be explained if we assume a large part of As exists in the apoplastic space rather than the symplast. The cell wall may contribute to trap apoplastic As(III), which accounts for the lack of a drastic reduction in As(III) content in nip1;1 mutant plants as well as nip1;2 and nip5;1 plants. It is also intriguing that As(III) tolerance was observed in nip1;1 mutant plants, while the reduction of As(III) contents in nip1;1 mutant plants is 30%. As(III) tolerance was not observed in nip1;2, nip5;1, nip6;1, and nip7;1 (Fig. 2A and supplemental Fig. S1) grown with 15 μm As(III), although all of them have the ability to transport As(III) (Refs. 18Bienert G.P. Thorsen M. Schüssler M.D. Nilsson H.R. Wagner A. Tamás M.J. Jahn T.P. BMC Biol.. 2008; 6: 26Google Scholar and 19Isayenkov S.V. Maathuis F.J. FEBS Lett.. 2008; 14: 1625-1628Google Scholar; Fig. 3B). Furthermore, a T-DNA insertion mutant of NIP5;1, which is expressed in roots and is localized to the plasma membrane (21Takano J. Wada M. Ludewig U. Schaaf G. von Wirén N. Fujiwara T. Plant Cell.. 2006; 18: 1498-1509Google Scholar), did not show As(III) tolerance (Fig. 2A). The expression level of NIP5;1 in roots was higher than that of NIP1;1 according to the MPSS data base. These results suggest that the decrease in As(III) uptake is not the only reason for the high As(III) tolerance observed in nip1;1 mutant plants. One possible reason for the lack of As(III) tolerance in nip1;2 and nip5;1 is due to the cell type specificity of expression. NIP1;1 might be expressed in cells that are directly or indirectly important for root growth, where the expression levels of other NIP genes are not detected or very low. Actually, the expression pattern of NIP5;1 is in contrast to that of NIP1;1. NIP5;1 is expressed in epidermal, cortical, and endodermal cell, but weakly in stele (21Takano J. Wada M. Ludewig U. Schaaf G. von Wirén N. Fujiwara T. Plant Cell.. 2006; 18: 1498-1509Google Scholar). Another possible explanation is that the disruption of NIP1;1 causes an altered distribution of of physiological substrate in roots, which may lead to As(III) tolerance through some unidentified mechanisms. One candidate substrate is glycerol. It has been shown that NIP1;1 is able to transport glycerol in yeast (31Weig A.R. Jakob C. FEBS Lett.. 2000; 481: 293-298Google Scholar). Physiological functions of glycerol in plants are less understood, but it has been suggested that glycerol is a component of cutin and suberin (32Pollard M. Beisson F. Li Y. Ohlrogge J.B. Trends Plant Sci.. 2008; 13: 236-246Google Scholar), which are hydrophobic cell wall barriers. Although the thickening of suberin layer in nip1;1 mutant plants was not evident (data not shown), disruption of NIP1;1 may change a cell wall property and may lead to the prevention of As(III) influx into roots. In conclusion, we have identified the key gene that determines As(III) tolerance in roots. Aquaglyceroporins probably exist in all plant species and have As(III) transport ability, because the As(III) transport activity have so far been observed in all aquaglyceroporins, regardless of species. In the course of writing this report, Ma et al. (17Ma J.F. Yamaji N. Mitani N. Xu X.Y. Su Y.H. McGrath S.P. Zhao F.J. Proc. Natl. Acad. Sci. U. S. A.. 2008; 105: 9931-9935Google Scholar) showed that the silicic acid transporter Lsi1 (OsNIP2;1) mediates As(III) uptake from soils and that disruption of each gene reduces the As content in plants, although its contribution to As accumulation in rice grains is small. NIP genes could be used as a molecular marker in crop breeding. Although the mechanisms of As(III) tolerance are not clear, a semidominant allele is useful for agricultural applications. F1 hybrid crops, including rice, are often used to boost yields. If similar semidominant alleles for As(III) tolerance are found in crops, then they can be directly used to produce As-tolerant hybrid crops. We thank the ABRC for providing materials used in this study. We also thank Emiko Yokota for technical assistance and Koji Kasai, Kyoko Miwa, Fabien Lombardo, and Islam Md. Rafiqul for critical reading of the manuscript.